Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in factor VIII. Factor VIII circulates as a metal ion-dependent heterodimer, comprised of a heavy (HC) and light (LC) chain. The dependence on metal ions for structure and activity remains poorly understood. We have demonstrated differential effects of Ca2+ and Cu+/2+ on HC and LC affinity and the generation of cofactor activity. Aim I studies will further probe these interactions focusing on sites for Ca2+ binding in LC and mechanisms by which Ca2+ modulates function. We have demonstrated that Cu2+ enhances factor VIII inter-chain affinity. The site for Cu occupancy is controversial. We will determine the residues involved in this coordination using a novel, metal ion-catalyzed oxidation technique specific for the Cu-coordinating residues. These studies will be complemented by Cu affinity and inter-chain stability analyses following factor VIII reconstitution using HC and LC containing relevant point mutations. The active cofactor, factor VIlla, is labile due to a weak affinity of the HC-derived A2 subunit for the A1/A3-C1-C2 dimer, and its dissociation represents the primary mechanism for cofactor inactivation. Sites involved in A2 subunit retention remain poorly characterized. Furthermore, the homology-modeled structure appears to conflict with several experimental observations. We have shown that approximately 90% of the thermodynamic stability for A2 subunit in factor VIlla is derived from interactions with the A1 subunit. In Aim II we will evaluate the interface of A1 and A2 subunits, as well as the A1/A3-C1-C2 interface, using chemical modification at selected residues coupled with mass spectrometry. Complementary approaches to assess A2 interactions will assess the affinity of relevant mutant A2 subunits, expressed in high yield in baculovirus, for the A1/A3-C1-C2 dimer using FRET and functional assays. An essential step in factor VIII activation is cleavage at Arg372 splitting the contiguous A1-A2 domains of HC into separate subunits. In Aim III we will evaluate a model for the interaction of thrombin with factor VIII HC. Identification and characterization of thrombin-binding sites in the A1 and A2 domains using FRET and solid-phase binding assays will determine the contributions of these sites to catalysis. Novel reagents including point mutations in factor VIII and thrombin anion-binding exosite mutants will be employed for binding and kinetic studies. We will also assess the influence of P3-P3'residues on cleavage at the Arg372 site based upon disparate rates for cleavage at the A1-A2 and A2-B junctions in HC. Definition of these issues will yield valuable structural and mechanistic insights into the biochemistry of the native as well as dysfunctional factor VIII molecules, and provide information that may be directly applied to the design of superior therapeutics for the treatment of hemophilia A.